Occurrence of arbovirus infections in two riverine populations in the municipality of Humaitá, Amazonas, Brazil

ABSTRACT Background: The riverine communities of the Amazon comprise different social groups that inhabit the rural areas on the banks of rivers and lakes. Residents usually travel by river to rural and urban areas and are then exposed to urbanized diseases such as those caused by arbovirus infection. In Brazil, emerging diseases such as dengue, Zika, chikungunya, and those caused by infection with Oropouche and Mayaro viruses necessitate epidemiological surveillance. This study was aimed at determining the frequency of positivity for immunoglobulin (Ig)G and IgM antibodies against Zika, chikungunya, and dengue viruses and performing molecular analyses to detect viral RNA for the Zika, chikungunya, dengue virus, Oropouche, and Mayaro viruses, in the same serum samples obtained from riverside populations. Methods: This cross-sectional study was conducted in a riverside population in the Humaitá municipality of the Brazilian Amazon. More than 80% of the local population participated in this study. Entomological samples were collected to identify local mosquito vectors. Results: Analysis of 205 human serological samples revealed IgG antibodies against the dengue virus in 85 individuals. No molecular positivity was observed in human samples. Entomological analyses revealed 3,187 Diptera species, with Mansonia being the most frequent genus. Aedes aegypti and Aedes albopictus were not detected in the two collections. Conclusions: IgG antibodies against the dengue virus were highly prevalent, suggesting previous exposure. The absence of the arbovirus vectors Aedes aegypti and Aedes albopictus in the samples supports the hypothesis that the infections recorded likely occurred outside the riverside communities investigated.


INTRODUCTION
The Amazon basin spans more than 7 million square kilometers, of which approximately 5 million square kilometers make up the Brazilian Amazon.It is an extensive area comprising forests and rivers and has a low demographic density, and the populations living in rural areas of the square kilometers suffer from isolation and difficulty in accessing public health services 1,2 .
The riverine communities of the Brazilian Amazon comprise different social groups, which include indigenous people, quilombolas, and migrants from other regions of the country and other countries.These populations live in rural areas along the banks of rivers and lakes of the Brazilian Amazon.Fishing, hunting, family farming, and plant and mineral extraction are their main means of income for these populations, in addition to subsidies from the federal government.They generally lack basic sanitation resources and are dependent on travel to urban areas for health care and acquisition of goods 3,4 .Travel to urban areas is usually by water and in small boats.These trips result in considerable costs for these riverine populations and are carried out only if and when there is a real need.This contact with urban areas creates opportunities for the acquisition of urbanized diseases.In the case of some diseases, however, riverine populations present with symptoms after returning to their communities, where they do not have adequate health care coverage 5,6 .
Among the main urbanized diseases in these populations are infections with arboviruses.Arboviruses are transmitted to humans by arthropods, specifically through the bite of hematophagous insects.They belong to the families Flaviviridae, Togaviridae, Bunyaviridae, Rhabdoviridae, and Reoviridae.There are more than 140 different arboviruses that infect humans and, of these, the most important include the dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus, West Nile virus, Mayaro virus (MAYV), and Oropouche virus (OROV).In Brazil, diseases caused by infection with the DENV, ZIKV, and CHIKV are currently considered emerging diseases [7][8][9] , and all three viruses are transmitted mainly by Aedes aegypti, which has a preference for environments close to human habitats and deposits its eggs in standing water (clean or slightly polluted).In favorable climatic conditions, mosquitoes show greater longevity, which allows females to increase their feeding and egg laying 10,11 .
Chikungunya, dengue, and Zika rarely cause death; however, they often lead to many debilitating conditions.For example, the symptoms associated with CHIKV infection can be chronic and may last for years 12 .Owing to rapid climate change, deforestation, population migration, disorderly occupation of urban areas, globalization, international travel, and social and economic factors and the resultant lack of basic sanitation, arboviruses represent a major and constant threat to tropical populations 13 .Therefore, surveillance of arboviruses, especially in regions with difficult access to health services and quality treatment, is necessary to identify future threats to these populations, and accordingly develop prevention and health promotion strategies to meet local needs.This study was aimed at determining the frequency of positivity for immunoglobulin (Ig)G and IgM antibodies against ZIKV, CHIKV, and DENV in samples obtained from riverine populations and conducting molecular analyses for viral RNA research for ZIKV, CHIKV, DENV, OROV, and MAYV, in addition to trying to detect the presence of Aedes aegypti and Aedes albopictus in riverine regions.

• Study type
This cross-sectional study involved the analysis of serological samples for the detection of IgG and IgM antibodies against DENV, ZIKV, and CHIKV and molecular analysis for viral RNA research.Samples were collected during health care initiatives in 2019 and 2020 for routine biochemical tests in two riverine communities in the state of Amazonas.At the time, the patients signed an informed consent form authorizing the storage and use of leftover samples for future research projects.These samples are stored in the biorepository of the Institute of Biomedical Sciences of USP-5 (Rondônia) and the clinico-epidemiological data are stored in the Red Cap database.In addition, entomological samples were collected twice from these communities, during the rainy and dry seasons, to detect the presence of arbovirus vectors.

• Study population
The riverine communities investigated in this study are part of the municipality of Humaitá, Amazonas, and are located on the banks of the Madeira River.The two communities included the Carará community (6°44'40.6"S 62°30'16.0"W) and the Espírito Santo community (6°46'10.6"S 62°27'30.6"W), both of which are in rural areas, 7 hours by boat from the nearest city.Access to the communities is by river only.The total population of the two communities is approximately 250 individuals, and the study included children over 3 years of age, adults, and the elderly.Their data and samples were collected during local health service expeditions.Owing to the low number of residents in these communities, the sample size was chosen for convenience.Samples that did not have enough material to perform the tests used in the study or for which complete records were not available in the Red Cap database were discarded.

• Ethical approval
This study was conducted according to the ethical principles of human research and was approved by the research ethics committee (CAAE no.51511415.4.0000.0013).The informed consent form was applied to both adults and minors, along with the minor assent form.Authorization for minors was granted by their legal guardians.

• Capture of vectors
To capture culicid eggs and larvae, oviposition traps, known as "ovitrampa" were used.This capture method was first described by Fay and Perry 14 .To capture adult specimens, different techniques were used, such as Shannon's technique, CDC-HP light trap (close to the ground and in tree canopy), and the Nasci aspirator [15][16][17] .The traps were installed in the forest, in areas surrounding the forest, and in rural homes.A total of 27 ovitraps were set up for each expedition, along with 14 CDC-HP light traps, the latter being distributed in different locations throughout the expedition period.For the entomological identification of culicids, genus and species were determined using identification keys 18,19 .

• Laboratory tests
To detect IgG and IgM antibodies against DENV, ZIKV, and CHIKV, the TR DPP ® ZDC IgM/IgG immunochromatographic test (Bio-Manguinhos, Rio de Janeiro, Brazil) was used, which uses a dual resource platform.The DPP ® Micro Reader reads the results electronically, thus eliminating possible errors of interpretation by human reading.It also enables automatic recording of the results and computer processing of the data.
Viral RNA extractions from human serum were performed in the Laboratory of Clinical and Molecular Virology (LVCM) at Instituto de Ciências Biomédicas, Universidade de São Paulo, by using a magnetic particle processor (MagMAX™ Express, Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.The extracted samples were then stored in a freezer at -80ºC until they were used for analysis.For molecular analysis of DENV, ZIKV, and CHIKV, quantitative reverse transcription  polymerase chain reaction (RT-qPCR) was performed using the EM8 reagent, which is a PCR mix containing reverse transcriptase, polymerase enzymes and nucleotides and magnesium from the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay kit (RV10259X; Seegene).For the PCR reaction, a concentration of 10 pM was used for each primer and probe.For molecular analysis of OROV and MAYV, conventional PCR was employed using the QuantFast SYBR Green RT-PCR kit (204156; Qiagen).The primers used are listed in Table 1.

• Statistical analysis
For statistical analysis, the programs Jamovi (version 2.3.21) and Microsoft Excel (version 14.0, Office 365) were used.Clinical and epidemiological data are stored in the Red Cap database.Associations were made between these data and the results of immunochromatographic tests.The Chi-square test was used, and the Fisher test was chosen for small samples.The level of statistical significance was <5% (p < 0.05).

• Serological analyses
Of the 212 samples available for the study, seven were excluded owing to a lack of sociodemographic data or lack of serum for performing the immunochromatographic tests.Thus, 205 patients were included in the study.Sociodemographic data are presented in Table 2.
According to the Brazilian Statute of Children and Adolescents, as per Law No. 8,069, of July 13 th , 1990, individuals up to the age of 12 years are considered children.Thus, this study included 64 children (31.3%) and 141 adults (68.7%) 24 .Most of the study participants were born and raised in the communities to which they belong (85.4%), and the rest were migrants from other cities in the state of Amazonas and neighboring states such as Rondônia and Acre.
The results in Table 3 show the socioeconomic correlations with the presence of antibodies, both IgM and IgG, against some of the viruses in this study.Indeterminate results were counted as negative in these analyses since it was not possible to confirm positivity.In the molecular analyses, none of the samples tested showed positivity for the genetic material of the viruses tested.

• Entomological analysis
A total of 3,187 Diptera species were identified in two entomological collections, the first in February 2022 during the rainy season and the second in July 2022 during the dry season.In all, 937 specimens were collected in the first collection and 2,250 in the second; the specimens belonged to 26 species and 11 genera of Culicidae (Table 4).Most of the specimens were female (98%).More than half of the specimens (62%) were not identified at a specific level because they were damaged and, consequently, did not possess the necessary morphological characters, or belonged to complexes of cryptic species of the genus Mansonia, currently under debate 25 .In fact, Mansonia was the predominant genus in the sampling performed using the CDC-HP light traps at the bases of trees and in the active search (64.3%, n = 2,051), followed by the genera Culex (n = 605) and Coquillettidia (n = 338).Specimens of Aedes spp.identified were not of Aedes aegypti or Aedes albopictus and were thus classified by genus only.
In the entomological collection techniques used in the study, which included the active search of breeding sites, ovitraps and CDC-HP light traps were placed at tree bases.The ones with the highest productivity in terms of the number of specimens caught were the CDC-HP light traps placed at tree bases; however, some specimens of typically wild genera, such as Sabethes and Wyeomyia, were collected only by the active searching of breeding sites and by using ovitraps.

DISCUSSION
This study showed a high seroprevalence for the arboviruses tested, which demonstrates that the study population has contact with diseases caused by arbovirus infection and is susceptible to the development of problems that these viruses can cause.Studies focused on rural populations of the Amazon are rare, and we believe that this is the first study on the seroprevalence of arboviruses in riverine populations of the Amazon to date.Studies in Malaysia have shown that arboviruses, especially the DENV, are no longer confined to urban areas because similar rates of seroprevalence have been recorded in urban and rural areas 26,27   The most frequent seropositivity in our study was for the DENV, corroborating the findings of the study by Júnior et al. 28 , which states that dengue is a neglected tropical disease whose burden has increased the most in recent decades in Brazil.The authors estimated that in 2016, dengue accounted for more than 92,000 disability-adjusted life years (DALYs) in the country, with a rate of 44.87 DALYs per 100,000 inhabitants.
In Brazil, the dengue surveillance system is based on passive notification of health care centers via laboratory diagnosis.This surveillance method may underestimate the incidence of dengue owing to underreporting of symptomatic and asymptomatic cases 29 .Between the years 2018 and 2021, 449 cases of dengue were registered in the municipality of Humaitá, which is the closest urban health care support center for the study population.Interestingly, in 2020, no cases were registered, which is most likely related to the coronavirus disease 2019 pandemic 30 .However, according to DATASUS 30 , in the period from 2018 to 2021, cases of infection were more frequent in the age group of 15 to 59 years.This corresponds to 373 cases of the 449 registered (83.07%), similar to the number of adults infected by any one of the arboviruses in this study (86.26%).
Martins et al. 31 conducted a study of the seroprevalence of arboviruses in Amazonian children in Acre and found that most children with detectable antibodies against the DENV were not clinically diagnosed by a health professional and had symptoms detected by their family and that, in fact, the number of children who with dengue may be up to 10 times higher than the numbers registered in the digital health system.This fact is justified by the difficulty of clinically diagnosing arboviral infections in children, since this age group presents with several febrile diseases and no specific symptoms, often because children do not know how to report them.This further increases the importance of serological diagnosis and the study of seroprevalence in populations to determine the true picture of infections 29 .
Although many studies affirm that women are at a higher risk for arbovirus infection, since they spend longer periods at home, considering the domestic habits of the Aedes aegypti vector 32,33 , this study demonstrated a higher prevalence of previous infections with the DENV in men (p < 0.018) (Table 5).This can be justified by the fact that infections did not occur in rural households but in urban areas, with trips to the local towns being mostly undertaken by men and, for cultural reasons, women remaining at home to perform chores and take care of children.One study conducted in a settlement in the rural area of the state of Acre in the Brazilian Amazon, also demonstrated a higher prevalence of arboviral infections in men.This was justified by their migratory activities to urban areas, which corroborates the results of our study 34 .
On comparing infections with any type of arbovirus among various age groups, via statistical analyses, we found a higher  prevalence of infection among adults.This finding reinforces the hypothesis that infections occur in urban areas, and because children travel less to cities owing to travel costs, there is lower exposure and lower seroprevalence.It is important to emphasize that most of the patients in the study were born in the communities investigated (85%); therefore, they probably acquired these arboviral infections when visiting urban areas.
Infection by the CHIKV was more significant compared with ZIKV infections in this study population.Research has shown that infections with CHIKV resulted in decompensation associated with pre-existing diseases, such as heart, kidney, and liver diseases; diabetes; hypertension; and systemic lupus erythematosus.Furthermore, the need for hospitalization owing to CHIKV infection is related to the exacerbation of pre-existing chronic non-communicable diseases (NCDs) 35 .In 2022, Pereira et al. 36 conducted a study of chronic NCDs in the same population as the present study and found a high prevalence of these diseases, which resulted in an even greater alert regarding the exposure of this population to this virus.
Although infections with MAYV and OROV are highly frequent infections in the Amazon region [37][38][39][40] , we did not find positivity of these viruses in molecular tests during the research of the genetic material of these viruses in the samples tested.Owing to limitations of the tests to identify antibodies against these viruses, only molecular analyses were performed.
Sanitary vigilance is important in riverine populations because, when individuals from these populations go into the forest for labor and cultural activities, they may get exposed to wild viral species.Other factors, such as intensification of agriculture, livestock farming, mining, and the installation of large hydroelectric dams, are known to increase deforestation and are associated with human mobility and density.These factors change the ecological patterns of virus-vectorhost interactions and result in a scenario that is totally favorable to the spread of diseases such as dengue, Zika, and chikungunya 41,42 .
No Aedes aegypti and Aedes albopictus specimens were identified in the entomological collections in this study.This is in contrast with the study of Sacramento et al. 43 , in which they performed seroprevalence tests for arboviruses and entomological surveillance in a village in the state of Ceará and identified the presence of mosquitoes in all the houses of the village under study.This fact reinforces the hypothesis that the infections recorded in our study occurred outside the riverine communities, more specifically in urban areas, or that another vector assumed the role of vectorization for these diseases.Molecular studies of the captured mosquitoes are underway (FIOCRUZ -RO) to evaluate the possibility of potential vectors for these arboviruses, since several studies have proven the capacity of other vectors for the transmission of DENV, ZIKV, and CHIKV [44][45][46][47][48] .
Although the tests used in this study have high sensitivity and specificity compared with those of other methods such as enzyme-linked immunosorbent assay, one of the limitations of the present study is that it was not possible to perform an assay to distinguish between the different dengue serotypes and confirm or rule out cross-reactivity.Another limitation is that sampling was performed by convenience (non-randomized), although it did account for 85% of the estimated population.
In Brazil, the arbovirus control model remains traditionally limited to specific investments in campaigns against mosquitoes and guidance through the media, with little effective impact on the fight against these diseases in general.There is a lack of innovation to address these endemic diseases, and there is a need for improvement in the training of health professionals, elimination of breeding sites, and health education initiatives for the general population.A study conducted in Icaraí-Caucaia (state of Ceará) on health education for dengue prevention and control concluded that health education initiatives were conducted ineffectively, without dialogue between health professionals and the general public 49 .Therefore, knowledge about these arboviruses and the ways to manage infections with them must be spread to remote communities, which do not know much about these pathogens, so that the population knows how to recognize the signs and symptoms of any complications that may occur and seek appropriate medical assistance.
The data presented suggest it is possible to highlight the importance of health education in this population and community health workers to enable them to manage infections with arboviruses and recognize symptoms.When communities get sick, diseases may aggravate, and at present, there is no medical assistance available for the necessary interventions in the short (severe dengue), medium (prenatal ZIKV infection) and long term (CHIKV-related arthrosis).

TABLE 2 :
Sociodemographic data of the study participants.

TABLE 3 :
Correlation of seroprevalence for the tested arboviruses and socioeconomic conditions.

TABLE 4 :
Diptera species collected in the Espírito Santo and Carará riverine communities (Amazonas state) according to sampling technique during the period from February 13 th to 17 th , 2022, and July 17 th to 23 rd , 2022, and their species/taxon and sex.

TABLE 5 :
Presence of IgM and IgG antibodies against dengue, Zika, and chikungunya viruses in the study population according to the biological sex.